Outline

Objective: Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder, characterized by a loss of GABA-ergic medium-sized spiny neurons in the caudate putamen. Neural progenitor cell (NPC) transplantation has emerged as a promising experimental therapeutic strategy for HD, however, the variables responsible for the success of this approach, including the optimal developmental stage of the grafted cells, are largely unknown. The creatine (Cr) kinase phosphotransfer system plays a pivotal role in cells with high and fluctuating energy demands, including NPCs. Supporting cellular energy metabolism by Cr supplementation is a clinically translatable method for improving cell transplantation strategies for HD. The present study aims at investigating possible effects on survival and differentiation of rat striatal NPCs at early (E14) and late (E18) developmental stages.

Methods: Striatal NPCs were isolated from E14 and E18 rat embryos and cultured for 7 days with and without Cr added at a concentration of 5 mM to the culture medium. NPC survival and GABA-ergic differentiation was assessed using immunocytochemistry, [3H]GABA uptake measurements and a 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay. Cr-mediated neuroprotection was investigated during a metabolic insult induced by serum and glucose deprivation.

Results: Chronic Cr treatment resulted in a significantly increased percentage of GABA-immunoreactive neurons as compared to untreated controls, both in the E14 (170.44Â±4.67) and E18 group (141.73Â±8.29). This effect was greater in E14 cultures (p<0.01). Short-term treatment from day in vitro (DIV) 6–7 also resulted in an increased induction (p<0.05) of the GABA-ergic phenotype in E14 (162.97Â±10.37), as compared to E18 cells (133.27Â±9.51). Short-term Cr treatment resulted in an increased GABA-uptake in both groups, but the effect did not differ between E14 and E18 cultures (p>0.05). Total neuronal cell numbers and general viability, as assessed with the MTT assay, were not affected (p>0.05). Protective effects of Cr against a metabolic insult were equal in E14 and E18 cultures (p>0.05).

Conclusions: Our findings demonstrate that the role of Cr as a GABA-ergic differentiation factor depends on the developmental stage of striatal NPCs, while Cr-mediated neuroprotection is not significantly influenced by this parameter. These findings may have implications for optimizing cell replacement strategies in HD.